Chondroitinase ABC (ChABC) is an enzyme that catalyzes chemical reactions involving the eliminative degradation of polysaccharides containing 1,4-beta-D-hexosaminyl and 1,3-beta-D-glucuronosyl or 1,3-alpha-L-iduronosyl linkages to disaccharides containing 4-deoxy-beta-D-gluc-4-enuronosyl groups. In animal tissues, ChABC acts on chondroitin 4-sulfate, chondroitin 6-sulfate and dermatan sulfate. Chondroitin sulfate is the most abundant of the glycosaminoglycans in biological systems and includes a variety of sulfated and non-sulfated carbohydrate derivatives including sulfated glucuronic acid, N-acetylgalactosamine, and iduronic acid. These materials are often linked to proteins to form chondroitin sulfate proteoglycans (CSPG), which play a number of structural and functional roles in biological systems (e.g., cell-cell interactions with extracellular matrix, directing or inhibiting neurodevelopment, compositing cartilage to become a structural component). The biosynthesis of these CSPG molecules and the proper location are critical to the normal functioning of humans. In fact, many diseases associated with the lack of CSPG, accumulation, or dislocation, such as diffuse Lewy body disease, mucopolysaccharide disease IV-A type and mucopolysaccharide disease VII type, disc herniation and so on.
Chondroitinase enzymes are a group of enzymes that can be divided into four types with different activity and substrate specificity (chondroitinase ABC, chondroitinase AC, chondroitinase B and chondroitinase C). These enzymes, particularly chondroitinase ABC, have recently been shown to be potential new biological agents for the treatment of many CSPG-related diseases. For example, researchers have found that ChABC can significantly promote functional recovery of a damaged spinal cord. Other researchers have shown that the injection of chondroitin sulfate into the affected parts of keloid and/or hypertrophic scars can greatly improve the symptoms. There has also been developed a successful model for the treatment of rabbit disc herniation with ChABC. Recent scientific studies have suggested that ChABC has more potential medical applications, including the treatment of amblyopia, nerve and spinal cord injuries, posterior vitreous detachment and inhibition of tumor metastasis.
Whether ChABC can be successfully used as a biotherapeutic agent is significantly dependent on the production and purification of ChABC. ChABC's existing upstream production techniques are based on the fermentation of unmodified proteus vulgaris or recombinant expression hosts (e.g., E. coli). By appropriate optimization, both of these production methods can yield a large number of non-purified matrices containing ChABC enzyme with a host organism. Because the matrices contain substantial quantities of protein, nucleic acid, endotoxin, and other impurities, the development of efficient downstream purification methods for obtaining high purity ChABC is critical. Further, the amount and nature of impurities from different expression hosts are very different, so in order to purify ChABC from a particular expression host, a specific downstream purification procedure will typically be developed separately, especially when using only non-specific interaction ion exchange (IEX) or hydrophobic interaction (HIC) chromatography. However, the development and optimization of IEX/HIC chromatography is time-consuming and requires considerable manpower and material resources. Consequently, the typical process requires multiple IEX/HIC chromatography steps to achieve high purity target proteins resulting in high overall cost.
In contrast to IEX/HIC chromatography, affinity chromatography presents several potential advantages. In affinity chromatography, molecules or molecules that interact specifically with the target protein are partially immobilized on the chromatography filler. This highly selective interaction can effectively extract the desired protein and remove impurities. However, affinity chromatography has not been used as a purification tool for ChABC without affinity tag. Because ChABC is an enzyme, it is not easy to design affinity chromatography for native ChABC. This is because most of the known molecules that can specifically bind to native ChABC are ChABC substrates that will be degraded when combined with the ChABC enzyme. In other words, if these substrate molecules are immobilized on the stationary phase, they will be decomposed by the enzyme during the purification process, so that ChABC cannot be captured from the solution.
Thus there is a need in the art for novel purification techniques for ChABC to enable cost-effective production of ChABC for use as a biotherapeutic drug.